Western Troubleshooting Help, please! - (Apr/23/2010 )

I have just started running Westerns again after a year's hiatus, and I am having a lot of trouble with the transfer. I have tried running the transfer at 25V for 70 minutes (on ice), which had a small amount of transfer, and running it t 90mV at 4 degrees C overnight, which appeared to shred my gel and set off an alarm because of evaporated methanol. I'm trying to look at VEGF (a protein at about 25kDa).

Any tips on the current/voltage to use?

I'm also wondering if my transfer buffer has too much methanol in it. I'm using 200 ml methanol, 100 ml 10X transfer buffer, and 700 ml DI water.

Thanks for all your help!

you could try 25V overnight.

keep the methanol the same.

if you are looking for high molecular weight proteins (>100 kDa) then you may want to add 0.05% sds to the transfer buffer.

mdfenko on Apr 23 2010, 12:13 PM said:

Use 10% MeOH and run the Western at 150 mA constant (~ 40 volts) for a minigel for 2 - 3 hrs. If you are using Biorad mini tanks then stick one of the ice resevoirs in the tank, put it on a stirrer plate and away you go. Works perfectly everytime for "everyday" WB. 20% MeOH is a little high for everyday use and is really not necessary. I use the following home made transfer buffer:

Glycine - 60 g
Tris - 6 g
MeOH - 200 mls
Make to 2L with H2O

Thats it, easy and saves you a load of cash.

bethashley on Apr 23 2010, 12:43 PM said:

hi,
if your transfer tank is biorad, try these.
1. methanol- 200 ml--- this is fine! should be 20%

2. transfer- 100v, 45-1 hr .. for such small protein, overnight is not good. 4 degree is important so as not to let it heat up. it´s important that you maintain the temp.. add ice box, do it in 4 degree room, if not do the transfer by surrounding ice.


cheers
kedar